Rotaviruses (RVs) are the most important cause of severe diarrhea in young children worldwide. These viruses also cause diarrheal disease in healthy adults, the elderly, and the immune compromised. RV replicates primarily in the mature villous tip cells of the small intestine. RVs infect most mammals in a host-specific fashion; in general, RV strains that cause infection in one mammal do not cause disease in another species. Natural RV infection and live attenuated vaccination effectively induces heterotypic (as well as homotypic) protection from symptomatic re-infection despite the existence of great serotype diversity among circulating RV strains. B cells and RV-specific antibodies mediate this immunity to symptomatic re-infection. Neither the RV target of humoral heterotypic immunity nor the mechanism that results in broadly cross-reactive immunity in people are currently well understood. To address these two unknowns, we propose to study a library of human intestine origin RV monoclonal antibodies (mAbs) with the following Specific Aims: Aim 1) Identify the targets of heterotypic and homotypic neutralizing human antibodies on the rotavirus virion at the protein, epitope, and structural levels. We will expand and extend our initial studies of the binding and traditional serologic neutralization phenotypes of human intestine-derived anti RV mAbs. Phenotyping will include novel neutralization assays employing primary human intestinal epithelial cell (IECs) small bowel enteroids. We will further extend these studies to identify, at the atomic level, the structural interaction between these functionally important mAbs and the three target RV surfaces proteins. Aim 2) Determine the mechanisms by which human mAbs to RV proteins VP8*, VP5*, and VP7 neutralize RV infection in a novel human small intestinal enteroid culture system. We will identify the mechanism by which these mAbs suppress (neutralize) RV replication using a variety of traditional and novel technical approaches to assess the ability of the mAbs to inhibit RV binding to human IECs, to inhibit cell entry, and to inhibit viral decapsidation. Aim 3) Use the human mAbs that most efficiently inhibit the binding of human rotaviruses to human intestinal epithelial cells to characterize the RV cell surface receptor on human intestinal cells. We will exploit the novel observation that many human mAbs targeting RV VP8* efficiently neutralize human RVs in human enteroid cultures and in HT-29 cells but not in MA104 cells. This finding suggests different RV receptor usage in human IECs vs. MA104 monkey kidney cells. We will use several genetic and biochemical characteristics of the putative RV receptor to better characterize/ identify the receptor that human RVs utilize to initially bind to human IECs. Aim 4). Use a selection of RV-specific neutralizing mAbs to VP8*, VP5*, and/or VP7 to design a competition-based serologic assay to better predict the level and effectiveness of humoral immunity to RV in people than current neutralization and/or IgA solid phase immunoassays. We will use selected RV neutralizing mAbs to develop a simple but predictive serological competition inhibition assay to measure epitope specific antibody responses to RV in naturally infected and/or RV vaccinated individuals. We will then correlate the level of competition inhibition of sera from infected and/or vaccinated individuals with previously obtained neutralization and /or RV binding titers from the same individuals to identify those single or combined mAbs for which competition binding activity provide the best correlate with neutralization titers and/or protected status from subsequent RV illness.